Method and apparatus for liposome production

ABSTRACT

A new method of producing liposomes is described using an in-line mixing system. The liposomes produced by this method find utility in numerous therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of 09/439,619 filed on Nov. 12, 1999now U.S. Pat. No. 6,534,018 and claims the benefit of U.S. ProvisionalApplication No. 60/108,355, filed on Nov. 13, 1998.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to a formulation for the delivery of avariety of beneficial and/or therapeutic compounds by encapsulationwithin liposomes, and a machine of unique design for the controlledproduction of same. Specifically the invention relates to a preciselycontrolled metering system for the mixing of the two or more componentsof the liposomal preparations so that the various factors affecting theconsistency, reproducibility and efficacy of the product may bemonitored and controlled. The present invention also relates to a methodand apparatus for the production of liposomal suspensions, emulsions,ointments and creams.

2. Background

Liposomes are lipid vesicles made of membrane-like lipid bilayersseparated by aqueous layers. Liposomes have been widely used toencapsulate biologically active agents for use as drug carriers sincewater- or lipid-soluble substances may be entrapped within the aqueouslayers or within the bilayers themselves. There are numerous variablesthat can be adjusted to optimize this drug delivery system. Theseinclude, the number of lipid layers, size, surface charge, lipidcomposition and the methods of preparation.

Liposomes have been utilized in numerous pharmaceutical applications,including injectable, inhalation, oral and topical formulations, andprovide advantages such as controlled or sustained release, enhanceddrug delivery, and reduced systemic side effects as a result of deliverylocalization.

Materials and procedures for forming liposomes are well-known to thoseskilled in the art and will only be briefly described herein. Upondispersion in an appropriate medium, a wide variety of phospholipidsswell, hydrate and form multilamellar concentric bilayer vesicles withlayers of aqueous media separating the lipid bilayers. These systems arereferred to as multilamellar liposomes or multilamellar lipid vesicles(“MLVs”) and have diameters within the range of 10 nm to 100 μm. TheseMLVs were first described by Bangham, et al., J. Mol. Biol. 13:238-252(1965). In general, lipids or lipophilic substances are dissolved in anorganic solvent. When the solvent is removed, such as under vacuum byrotary evaporation, the lipid residue forms a film on the wall of thecontainer. An aqueous solution that typically contains electrolytes orhydrophilic biologically active materials is then added to the film.Large MLVs are produced upon agitation. When smaller MLVs are desired,the larger vesicles are subjected to sonication, sequential filtrationthrough filters with decreasing pore size or reduced by other forms ofmechanical shearing. There are also techniques by which MLVs can bereduced both in size and in number of lamellae, for example, bypressurized extrusion (Barenholz, et al., FEBS Lett. 99:210-214 (1979)).

Liposomes can also take the form of unilamellar vesicles, which areprepared by more extensive sonication of MLVs, and consist of a singlespherical lipid bilayer surrounding an aqueous solution. Unilamellarvesicles (“ULVs”) can be small, having diameters within the range of 20to 200 nm, while larger ULVs can have diameters within the range of 200nm to 2 μm. There are several well-known techniques for makingunilamellar vesicles. In Papahadjopoulos, et al., Biochim et BiophysActa 135:624-238 (1968), sonication of an aqueous dispersion ofphospholipids produces small ULVs having a lipid bilayer surrounding anaqueous solution. Schneider, U.S. Pat. No. 4,089,801 describes theformation of liposome precursors by ultrasonication, followed by theaddition of an aqueous medium containing amphiphilic compounds andcentrifugation to form a biomolecular lipid layer system.

Small ULVs can also be prepared by the ethanol injection techniquedescribed by Batzri, et al., Biochim et Biophys Acta 298:1015-1019(1973) and the ether injection technique of Deamer, et al., Biochim etBiophys Acta 443:629-634 (1976). These methods involve the rapidinjection of an organic solution of lipids into a buffer solution, whichresults in the rapid formation of unilamellar liposomes. Anothertechnique for making ULVs is taught by Weder, et al. in “LiposomeTechnology”, ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol.1, Chapter 7, pg. 79-107 (1984). This detergent removal method involvessolubilizing the lipids and additives with detergents by agitation orsonication to produce the desired vesicles.

Papahadjopoulos, et al., U.S. Pat. No. 4,235,871, describes thepreparation of large ULVs by a reverse phase evaporation technique thatinvolves the formation of a water-in-oil emulsion of lipids in anorganic solvent and the drug to be encapsulated in an aqueous buffersolution. The organic solvent is removed under pressure to yield amixture which, upon agitation or dispersion in an aqueous media, isconverted to large ULVs. Suzuki et al., U.S. Pat. No. 4,016,100,describes another method of encapsulating agents in unilamellar vesiclesby freezing/thawing an aqueous phospholipid dispersion of the agent andlipids.

In addition to the MLVs and ULVs, liposomes can also be multivesicular.Described in Kim, et al., Biochim et Biophys Acta 728:339-348 (1983),these multivesicular liposomes are spherical and contain internalgranular structures. The outer membrane is a lipid bilayer and theinternal region contains small compartments separated by bilayer septum.Still yet another type of liposomes are oligolamellar vesicles (“OLVs”),which have a large center compartment surrounded by several peripherallipid layers. These vesicles, having a diameter of 2-15 μm, aredescribed in Callo, et al., Cryobiology 22(3):251-267 (1985).

Mezei, et al., U.S. Pat. Nos. 4,485,054 and 4,761,288 also describemethods of preparing lipid vesicles. More recently, Hsu, U.S. Pat. No.5,653,996 describes a method of preparing liposomes utilizingaerosolization and Yiournas, et al., U.S. Pat. No. 5,013,497 describes amethod for preparing liposomes utilizing a high velocity-shear mixingchamber. Methods are also described that use specific starting materialsto produce ULVs (Wallach, et al., U.S. Pat. No. 4,853,228) or OLVs(Wallach, U.S. Pat. Nos. 5,474,848 and 5,628,936).

A comprehensive review of all the aforementioned lipid vesicles andmethods for their preparation are described in “Liposome Technology”,ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II & III(1984). This and the aforementioned references describing various lipidvesicles suitable for use in the invention are incorporated herein byreference.

Current methods of manufacturing liposomes are typically batchprocesses. Attempts at large scale or continuous manufacturing havelargely been unsuccessful, primarily due to the problems associated withmixing an aqueous liquid phase with the lipid phase and the need tomaintain the lipid phase at a relatively constant temperature.

Accordingly, there is a need for an improved method for the productionof liposomes, preferably one that can produce liposomes in a continuousfashion rather than by batch methods, without the variations anduncontrolled differences which make large scale production of liposomalpreparations problematic. In addition, there is a need for an improvedmethod and apparatus for producing other liquid compositions, includingbut not limited to emulsions, ointments and creams. Those needs are metby the instant invention.

SUMMARY OF THE INVENTION

The present invention relates to a method for the continuous productionof a composition of matter, such as lipid vesicles, by in-line mixing,said method comprising: (a) preparing a first phase, such as a lipidphase, and storing the lipid phase in a first storage means that ismaintained at a set temperature; (b) preparing a second phase, such asan aqueous phase, and storing the aqueous phase in a second storagemeans that is maintained at a set temperature; (c) combining the lipidand aqueous phases by means of a mixing device having first and secondmetering systems, a pre-mixing system and a mixer, such as a staticmixer, by: transferring the lipid phase from the first storage means tothe first metering system by a first pressurized transfer means andtransferring the aqueous phase from the second storage means to thesecond metering system by a second pressurized transfer means;transferring the lipid phase from the first metering system to a firstinlet orifice in the pre-mixing system by a third pressurized transfermeans and transferring the aqueous phase from the second metering systemto a second inlet orifice in the pre-mixing system by a fourthpressurized transfer means; wherein the lipid phase and aqueous phasesare transferred to the pre-mixing system with a high velocity creatingturbulent flow; combining the lipid and aqueous phases in the pre-mixingsystem by shear mixing under conditions to insure that the lipid phasebecomes fully hydrated by the aqueous phase to form a pre-mixedformulation; and transferring the pre-mixed formulation from an outletorifice of the pre-mixing system to the mixer, such as by a fifthpressurized transfer means or other suitable connection or fitting; (d)forming a mixed formulation comprising lipid vesicles, in the mixer bycausing the pre-mixed formulation to traverse the mixer; (e) optionallymeasuring the optical properties of the lipid vesicles; and (f)dispensing the mixed formulation from the mixer into a storage chamber,into a means for further modification of the properties of the lipidvesicles, or into a means of packaging the mixed formulation.

In a second aspect, the invention relates to lipid vesicles and othercompositions of matter produced by the method of the invention.

In yet another aspect, the invention pertains to a method of producingcompositions such as lipid vesicles using an in-line mixing system,where an active agent is encapsulated in either the aqueous core of thelipid vesicles, within the lipid bilayer of the lipid vesicles, or both.In still another aspect, the invention relates to lipid vesicleencapsulated active agents produced by the method of the invention.

Another aspect of the invention pertains to an apparatus for thecontinuous production of a composition of matter such as lipid vesiclesby in-line mixing, said apparatus comprising: (a) a first phase, such asa lipid phase, storage means capable of being maintained at a settemperature and a first pressurized transfer means for transferring thelipid phase from the storage means; (b) a second phase, such as anaqueous phase, storage means capable of being maintained at a settemperature and a second pressurized transfer means for transferring theaqueous phase from the storage means; (c) a mixing device comprising: afirst metering system for receiving the lipid phase from the firstpressurized transfer means; a second metering system for receiving theaqueous phase from the second pressurized transfer means; a pre-mixingsystem for preparing a pre-mixed formulation, having a pre-mixingchamber; a third pressurized transfer means for transferring the lipidphase from the first metering system to a first inlet orifice in thepre-mixing system and a fourth pressurized transfer means fortransferring the aqueous phase from the second metering system to asecond inlet orifice in the pre-mixing system; a mixer, such as a staticmixer, for preparing a mixed formulation comprising lipid vesicles,having a mixing chamber and an optional means for determining theoptical properties of the mixed formulation; a fifth pressurizedtransfer means or other suitable connection or fitting for transferringthe pre-mixed formulation from the outlet orifice of the pre-mixingsystem to the mixing chamber; and an optional means for applyingultrasonic energy to the pre-mixing chamber, the mixing chamber or bothof said chambers; and (d) a dispensing means for transferring the mixedformulation from the mixing chamber into a storage chamber, or into ameans for further modification of the properties of the lipid vesiclesor into a means of packaging the mixed formulation.

In another aspect, the invention relates to liposomes produced by theapparatus of the invention.

Still another aspect of the invention relates to the use of the methodand apparatus described herein for the manufacture of emulsions, and theemulsions produced thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the invention.

FIG. 2 is cut-away side view of one embodiment of the invention.

FIG. 3 is a top view of the embodiment shown in FIG. 2.

FIG. 4 illustrates one embodiment of the metering system of theinvention.

FIG. 5 is a cross-sectional view of a metering pump, taken along line5—5 of FIG. 4.

FIG. 6 illustrates one embodiment of a manifold.

FIG. 7 is a cross-sectional view of a manifold, taken along line 7—7 ofFIG. 6.

FIG. 8 is a cross-sectional view of a manifold, taken along line 8—8 ofFIG. 7.

FIG. 9 is cut-away side view of another embodiment of the invention.

FIG. 10 is a top view of the embodiment shown in FIG. 9.

FIG. 11 is a partial cross-section of one embodiment of the pre-mixersystem.

FIG. 12 illustrates another embodiment of a manifold.

FIG. 13 illustrates the rear view of the manifold of FIG. 12.

FIG. 14 illustrates the front view of a precise metering pump thatengages the manifold of FIG. 12.

FIG. 15 is a cross-sectional view of a manifold, taken along line 15—15of FIG. 12.

FIG. 16 is a cross-sectional view of a manifold, taken along line 16—16of FIG. 12.

FIG. 17 is a cross-sectional view of a manifold, taken along line 17—17of FIG. 12.

FIGS. 18 and 19 illustrate control panels for the invention.

FIG. 20 illustrates the bottom view of a power distribution module.

DESCRIPTION OF THE INVENTION

The apparatus and method of the instant invention permits the flexibleadaptation of a number of methods of production of lipid vesicles andother compositions of matter in a small space with a single device thatcan utilize a variety of production methodologies and techniques asdictated by the particular requirements of the product formula, anyactive agent to be incorporated into the composition and the applicationfor which the final product is intended. For purposes of illustrationonly, the method and apparatus of the invention will be described forthe production of lipid vesicles. However, the product and othercompositions such as emulsions, ointments and creams are alsocontemplated by the invention.

Typically, an active agent is encapsulated in either the aqueous core ofliposomes, within the lipid bilayer of the liposomes, or both, bydissolving or dispersing the agent in a lipid-containing organicsolvent. However, the invention also contemplates the manufacture oflipid vesicles that do not contain any active agents. Such emptyliposomes are currently used in cosmetic preparations and may berequired as placebo material in clinical trials of therapeuticformulations. As used herein, the term “active agent” includesbiologically active agents and is used to mean any molecule that acts asa beneficial or therapeutic compound, when administered to an animal,such that it prevents or alleviates a disease, arrests or alleviates adisease state or treats a disease in an animal, particularly a mammal,more particularly a human, and includes: preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; inhibiting the disease, i.e.arresting its development; or relieving the disease, i.e. causingregression of the disease. Examples of active agents include by means ofillustration and not limitation, anti-inflammatory agents; anti-cancerand anti-tumor agents; anti-microbial and anti-viral agents, includingantibiotics; anti-parasitic agents; vasodilators; bronchodilators,anti-allergic and anti-asthmatic agents; peptides, proteins,glycoproteins, and lipoproteins; carbohydrates; receptors; growthfactors; hormones and steroids; neurotransmitters; analgesics andanesthetics; narcotics; catalysts and enzymes; vaccines; geneticmaterial such as DNA.

Although the products of the invention are particularly well suited forpharmaceutical use, they are not limited to that application, and may bedesigned for food use, agricultural use, for imaging applications, andso forth. Accordingly, the term “active agent” is more broadly used tomean any chemical or material that is desired to be applied,administered or used in a liposome formulation, and can include, by wayof illustration and not limitation, pesticides, herbicides, cosmeticagents and perfumes, food supplements including vitamins and minerals,flavorings, and other food additives, imaging agents, dyes, fluorescentmarkers, radiolabels, plasmids, vectors, viral particles, toxins,catalysts, and so forth. The term “payload” is used herein to includeactive agents as defined above, along with any other ingredients thatmay be desirable to add to the product such as, by way of illustrationand not limitation, diagnostic markers including radiolabels, dyes,chemiluminescent and fluorescent markers; contrasting media; imagingaids; and so forth. The payload can be a solid, liquid or gas.

Numerous phospholipids are useful in the manufacture of lipid vesicles,particularly those selected from the group consisting of phosphatidylchlolines, lysophosphatidyl chlolines, phosphatidyl serines,phosphatidyl ethanolamines, and phosphatidyl inositols. Particularlysuitable phospholipids are natural phospholipids such as soybean oilbased phospholipids, for example the phosphatidylcholines, Phospholipon®90H, 80H, 90G and 80G (American Lecithin Company, Oxford, Conn.), andLecinol (Nikken). The phospholipid can be modified using a modifyingagent such as a cholesterol, stearylamine or tocopherol. The solvent isthen evaporated, usually under reduced pressure, to yield a thin lipidfilm containing the active agent. The lipid film is then hydrated, withagitation, using an aqueous phase containing any desired electrolytesand lipid vesicles containing the active agent are produced.

The invention relates to a method and apparatus that produces a veryconsistent reproducible, continuous and continuously variable outputstream composed of liposomes in the form of multilamellar lipid vesicles(“MLVs”), unilamellar lipid vesicles (“ULVs”) or oligolamellar vesicles(“OLVs”) containing encapsulated active agents. As used herein, theterms “liposome” and lipid vesicle” are used interchangeably and areintended to include MLVs, ULVs and OLVs. The design of the equipmentdescribed herein is intended to allow constant monitoring of variousfactors that affect the size, distribution, structure, number and drugencapsulating efficiency of the vesicles so produced. The methods andembodiments described herein are typically capable of producingliposomal formulation at the rate of about 10 to 200 liters/hour,preferably 25 to 100 liters/hour. It is understood that these rates aremerely illustrative of the rates attainable by the method and apparatusdescribed herein. Lower rates may be desirable under certaincircumstances, and higher rates may be attainable by standardoptimization techniques, as are well known in the art, both of which areencompassed by the invention.

MLVs, ULVs and OLVs produced by state of the art methods tend to includea broad distribution of sizes and shapes as well as a broad range ofpayload volumes. Frequently, liposomal preparations include asubstantial proportion of non-payload carrying vesicles. The inventionconsists of several innovations intended to consistently andcontrollably monitor and optimize the production of payload carryingMLVs, ULVs and OLVs, i.e., provides for higher encapsulation efficiencythan state of the art methods with very reproducible results.

The instant invention pertain to an apparatus useful for the continuousproduction of a composition of matter by in-line mixing. In oneembodiment of the invention, the apparatus comprises a first phasestorage means capable of being maintained at a set temperature and afirst pressurized transfer means for transferring the first phase fromthe storage means, along with an second phase storage means capable ofbeing maintained at a set temperature and a second pressurized transfermeans for transferring the second phase from the storage means. In apreferred embodiment, the first phase is a lipid phase (optionallycontaining an active agent) and the second phase is an aqueous phase.The lipid phase storage means is capable of being maintained at a settemperature by a first temperature control means, typically within therange of about 20 to 75° C. Similarly, the aqueous phase storage meansis capable of being maintained at a set temperature by a secondtemperature control means, typically within the range of about 20 to 75°C. In one embodiment of the invention, the lipid phase and aqueous phasestorage means are equipped with a means for continuously replenishingthe lipid and aqueous phases. In this manner, the storage means functionas a temperature stabilization means such that the lipid and aqueousphases are continuously fed into the storage means, where thetemperature of each phase is stabilized prior to introduction intopressurized transfer means that exits each respective storage vessel.

The apparatus also has a mixing device that comprises a first meteringsystem for receiving the lipid phase from the first pressurized transfermeans, a second metering system for receiving the aqueous phase from thesecond pressurized transfer means, a pre-mixing system for preparing apre-mixed formulation, a third pressurized transfer means fortransferring the lipid phase from the first metering system to a firstinlet orifice in the pre-mixing system and a fourth pressurized transfermeans for transferring the aqueous phase from the second metering systemto a second inlet orifice in the pre-mixing system. The pre-mixingsystem comprises a pre-mixing chamber having a first and second inletorifice. In one embodiment of the invention, the pre-mixing systemfurther comprises a means for creating turbulence in the aqueous phaseprior to entry into the pre-mixing chamber.

The apparatus also has a mixer such as a static mixer for preparing amixed formulation comprising lipid vesicles, having a mixing chamber andan optional means for determining the optical properties of the mixedformulation, a fifth pressurized transfer means for transferring thepre-mixed formulation from the outlet orifice of the pre-mixing systemto the mixing chamber or other suitable connection or fitting; and anoptional means for applying ultrasonic energy to the pre-mixing system,the mixing chamber or both. In a preferred embodiment, the opticalproperties of the mixed formulations are measured, with the means fordetermining the optical properties of the mixed formulation beingconfigured so as to control the first and second temperature controlmeans and the first and second metering systems.

Typically, the means for determining the optical properties will be inthe form of an optical detector that consists of a light source, such asan incandescent bulb, light emitting diode or other suitable lightemitting apparatus, which is positioned on one side of a chamber withparallel transparent windows. A sensor such as a photocell,phototransistor or photoresistor is positioned on the other side of thechamber, opposite the light source. The purpose of the optical detectoris to generate a signal or value, such as resistance or voltage, thatwill vary in linear fashion in correlation to the opacity, translucencyor other optical properties of the product, which vary with time,temperature, concentration and flow rates of the lipid and aqueous phasecomponents. The signal thus derived, generated or measured, can then beused to activate transfer means, controls, pumps, motors, heaters, andso forth, through an interactive computer system, to maintain, alter, oradjust the properties of the product with greater precision. The signalcan also be used to control and direct product flow to either a storagechamber, a means for packaging or to a waste receptacle, if for example,the product failed to meet desired specifications. One embodiment of theoptical detector might include a diode that emits UV light that isabsorbed by a specific active agent. In this manner, when the activeagent is detected, the product flow is directed to a means forpackaging; if the active agent is absent or present in an unsuitableamount, the product flow can be directed to a waste receptacle or theoperation can be stopped so as to permit evaluation and correction ofany problems.

The apparatus and method of the invention provide for lipid phase andaqueous phase streams that are as pulse-less as possible and aremaintained at a constant pressure. This is a achieved by the precisemetering systems described herein, each of which is provided with a pumpthat operates under positive pressure and in such a manner so as toprovide precise volumetric delivery.

The mixer is preferably a static mixer, such as a laminar division typeinline mixer. The mixer may have a means for controlling the temperatureof the mixing chamber, which is typically within the range of about 20to 80° C. In addition, the mixer may also have a means for controllingthe degree and rate of mixing within the mixing chamber. The mixingdevice of the apparatus may also have a means for controlling thetemperature within the open space of the mixing device, which is alsotypically within the range of about 20 to 80° C.

Finally, the apparatus has a dispensing means for transferring the mixedformulation from the mixing chamber into a storage chamber. Thisembodiment of the apparatus is particularly useful for the production oflipid vesicles, and more particularly multilamellar lipid vesicles. Theapparatus of the invention is readily evaluated as to its particularsuitability for manufacturing lipid vesicles having a pre-specifiedcomposition and configuration. Typically, two measurements are utilizedto evaluate a method of manufacturing lipid vesicles: the encapsulatedmass, the amount of encapsulated material/amount of lipid (wtmaterial/wt lipid); and the captured volume, the amount of encapsulatedaqueous phase/amount of lipid in vesicle (vol. aqueous/wt lipid).

In another embodiment, the apparatus also has a means for homogenizationor sonication, which is located between the dispensing means and thestorage chamber. This latter embodiment is particularly useful for theproduction of unilamellar lipid vesicles.

The apparatus may also have additional storage means for additionalliquid phases such as a second lipid phase, a pre-mixed lipidphase-aqueous phase mixture, and/or a pre-formed lipid vesicle phase.

In operation, the apparatus of the invention typically operates underpressures within the range of about 10 to 90 psia, more commonly about40-80 psia. It is understood that the apparatus and method of theinvention are not necessarily operating under a constant pressure, andthe actual pressure will vary among the components of the apparatus. Thefluid flow rate of the lipid phase is usually about 3-200 cm³/sec, morecommonly 4 to 80 cm³/sec. The fluid flow rate of the aqueous phases isusually about 5-300 cm³/sec, more commonly about 10 to 100 cm³/sec. Thefluid flow rate at the various stages of the process and within thevarious components of the apparatus is determined by the initial flowrates, such that the flow rates of the lipid and aqueous phases remainconstant and the flow rate of the mixed streams will be cumulative ofthe incoming lipid and aqueous rates. The fluid flow rate of the lipidphase is typically slower than that of the aqueous phase and will dependupon the desired composition of the product, i.e., the mixedformulation, but will usually be about 20-30% that of the aqueous phase.So, for example, for an aqueous fluid flow rate of about 20 cm³/sec onemay select a lipid fluid flow rate of about 6 cm³/sec, which will thenprovide for a combined phase flow rate of about 26 cm³/sec.

The dispensing means of the apparatus may have a means for controllingthe rate at which the formulation is transferred from the mixing chamberinto the storage chamber, which may be part of a packaging machine. Thisrate controlling means maintains the rate at which the mixed formulationis transferred.

In yet another preferred embodiment of the invention, each meteringsystem contained within the apparatus has a precise metering pump and amanifold. Each pump and manifold has a plurality of inlet and outletmeans, where each pump inlet means communicates with a manifold outletmeans and each pump outlet means communicates with a manifold inletmeans, The manifold, along with having a plurality of inlet and outletmeans, also has a manifold outlet orifice and a manifold inlet orifice.In operation, the inlet orifice of a first manifold is in communicationwith the first pressurized transfer means and the outlet orifice of afirst manifold is in communication with the third pressurized transfermeans, the inlet orifice of a second manifold is in communication withthe second pressurized transfer means and the outlet orifice of a secondmanifold is in communication with the fourth pressurized transfer means.The term “in communication with” is intended to mean connections suchas: an inlet means that is configured so as to fit within an outletmeans (or vice versa), an inlet means that is positioned immediatelyadjacent to an outlet means, and an inlet means that is connected to anoutlet means by pipes, tubing or other suitable conduit that permitsfluid flow, and so forth.

The various embodiments of the method and apparatus of the invention arebest understood with reference to the Figures.

FIG. 1 is a schematic illustration of one embodiment of the apparatus ofthe invention for the production of compositions such as lipid vesicles,particularly MLVs, and also illustrates the method by which theliposomal formulation is produced. The apparatus 10 has individual meansfor storing each component of the formulation, each component beingstored at a set temperature. The storage means are illustrated in FIG. 1as vessels or reservoirs 12 and 14, which store the components of theformulation at specified temperatures, which are controlled bytemperature controls 16 and 18. For example, one vessel may contain thelipid or lipid like phase and the other vessel might contain an aqueousphase, either one of which may contain one or more active agents orother payload.

It is understood, however, that although only two vessels areillustrated, the invention is not limited to that number and any numberof storage means can be used, and the actual number will vary dependingupon the number of components in the formulation. For example, a thirdvessel or reservoir may be used to add an additional lipid phasecomponent to the formulation. Such an additional lipid phase would beadded, for example, to facilitate the incorporation of one or moredifferent, separately encapsulated active ingredients, or another formof phospholipid with a higher or lower melting point which would alterthe properties of the mixture and would be added concurrently with, inadvance of, or following the addition of the primary lipid phase to theaqueous phase in the pre-mixing system by means of an additionalmetering pump and at a point where it could be introduced either before,after or concurrently with the other streams. Similarly, an additionalvessel (and metering pump) may be used to add a previously preparedtwo-phase mixture, for example, to incorporated a second or third activeagent into the formulation, and would be added either concurrently with,in advance of, or following the addition of the primary lipid phase tothe aqueous phase in the pre-mixing chamber.

The storage means useful in the method of the invention are typicallyvessels, reservoirs or tanks of any suitable configuration and can bemade of any material that will withstand any temperature and pressurerequirements and will not react with the components stored therein.Typical materials include, by way of illustration and not limitation,stainless steel, glass, suitable plastics, fluoropolymers, etc. Thestorage means can be large enough to store a sufficient amount of thecomponents so as to enable production of a specified amount offormulation. On the other hand, the storage means 12 and 14 can functionas mid-stream storage vessels that are continuously being replenishedfrom an external source such as a larger vessel, not shown, as thecomponents are being dispensed. In that manner, the amount of componentstored within means 12 and 14 will remain relatively constant throughoutthe production cycle. The storage means may vary in size depending uponthe individual needs of the process being run, however, they willtypically hold from 0.5 to 15 liters, preferably from 1 to 1.5 liters,more preferably about 1.5 liters for a system that is not beingreplenished from an external source, and from 0.5 to 10 liters,preferably from 1 to 5 liters, more preferably about 5 liters for asystem that is being replenished from an external source.

As indicated above, it is desirable to maintain the components of theformulation at set temperatures, which are controlled by temperaturecontrols 16 and 18. These temperature controls can be commerciallyavailable discrete controllers as may be obtained from OmegaEngineering, Inc. (Stamford, Conn.), programs running on a computer, orladder type time, flow controlled controllers, and so forth.

The lipid and aqueous phases are typically maintained at a temperaturewithin the range of 20-80° C. However, it may be desirable to maintain aslightly higher temperature for the lipid phase component. For example,a preferable range for the lipid phase might be about 55 to 65° C., morepreferably about 60° C., while the corresponding temperature for theaqueous phase component would be within the range of about 50 to 60° C.,more preferably about 55° C. The optimal temperature differential willbe determined by the formulation itself and the desired productcharacteristics.

Each component is delivered to the mixing device 20 by pressurizedtransfer means 22 and 24, each of which is fitted with a precisemetering system 26 and 28 to control the amount of material transferredfrom the storage means to the mixing device. Each metering system wouldtypically comprise a pump, along with a manifold to control the pumpoutput and input in order to eliminate any pressure pulsations of thecomponent stream that would interfere with the precise mixing ratiosneeded for consistent product quality.

The mixing device 20 has a pre-mixing system such as a pre-mixer 29having a pre-mixing chamber 30, where the individual components, i.e.,the lipid phase and the aqueous phase are introduced under pressure bypressurized transfer means 23 and 25. Pressurized transfer means 22 and24 transfer the formulation components from their respective vessels 12and 14, to the precise metering systems 26 and 28. Pressurized transfermeans 23 and 25 then transfer the formulation to the pre-mixing chamber30. The mixing device may also be provided with a means, not shown, forestablishing a gradient between the two component streams.

It is critical that the lipid and aqueous phases be transferred to thepre-mixing system with sufficient velocities such that turbulent flow iscreated in the pre-mixing chamber so as to provide shear mixing and toinsure that the lipid phase becomes fully hydrated by the aqueous phase.For example, the lipid phase can be introduced to the aqueous phase bymeans of a concentrically centered hypodermic sized tube in the centerof the tube carrying the aqueous phase. The interface between the twophases is such that the friction between the inner lipid stream and theouter aqueous stream creates laminar turbulence and eddy currents thatwill initiate the interfacial mixing process.

Transfer means 22, 24, 23 and 25 are typically configured as flexibletubing, stainless steel tubing, or as channels in a block of suitablematerial, and can be made of any non-corrosive, non-reactive materialsincluding, by way of example and not limitation, plastic, rubber,aluminum, stainless steel, plastics, fluoropolymers such as Teflon andpolyvinylidene fluoride (PVDF), etc.

The pre-mixer 29 is used for preparing a pre-mixed formulation and isdesigned to create a turbulent vortex in one component stream into whichthe second component stream is injected via high pressure. The combinedcomponent streams are then transferred via transfer means 32 andintroduced into the mixer 33 having an in-line mixing chamber 34 of highshear, high pressure design. Transfer means 32 can be of a configurationand materials such as described above for transfer means 22, 24, 23 and25. Preferably transfer means 32 is a relatively short fitting thatserves to connect the outlet of the pre-mixer with inlet of the mixer ormay simply be the juncture of the pre-mixer outlet and mixer inlet suchas when the outlet of the pre-mixer is positioned adjacent to andcommunicates with the inlet of the mixer.

The length of the mixing chamber 34 can be varied to control the numberof laminar divisions that the stream passes through. The mixer 33 ispreferably a static mixer. As used herein the term “static mixer” isused to refer to a mixer whose internal chamber creates turbulence inthe fluid flow by the presence of, for example, a spiral or baffledinterior casing, such that movement of the component streams through themixer creates a mixed product, without the need for any moving parts inthe mixer. Suitable static mixers include laminar division type inlinemixers or an inline static ISG (Interfacial Surface Generator) mixingdevice, which has either a stream division or intercalated spiral baffledesign. Commercially available static mixers that are suitable for usein the instant invention include the TAH 70, 85, 100, 120 and 160 Seriesmotionless mixers sold by TAH Industries, Inc. (Robbinsville, N.J.), andthe ISG motionless mixer sold by Charles Ross & Son Company (Hauppauge,N.Y.).

The mixing chamber 34 is equipped with a means 36 for controlling thetemperature of the chamber. It is preferable to maintain the temperatureof the mixing chamber within the range of about 20 to 80° C., preferably50 to 70° C., more preferably about 60° C. The temperature controllingmeans 36 is typically a discrete thermocouple, a platinum thermocoupletype automatic controller, a ladder type controller, or a controlroutine operating on a computer. The mixing chamber 34 is also equippedwith a means 38 for controlling the degree and rate of mixing within thechamber. This mixing controlling means 38 is typically a means formoving the lipid and/or aqueous phase transfer means in and out of thechamber to adjust the insertion point for the desired degree ofturbulence. Also a means may be provided for adjusting the angle of thetransfer means relative to the wall of the mixing chamber to enhance ordecrease the rotational turbulence thereby induced.

After the formulation is mixed, it is dispensed via dispensing means 40,which is equipped with a control means 42 for controlling the rate atwhich the formulation is transferred from the mixing chamber 34 into thestorage chamber 44. The storage chamber can be part of a packagingmachine, not shown. The mixed formulation can be used as is, without theneed for any solvent removal.

Dispensing means 40 is typically configured as a sanitary valved outlet,and can be made of any non-corrosive, non-reactive materials including,by way of example and not limitation, plastic, rubber and aluminum.

The flow rate controlling means 42 typically operates by adjusting thestepping rate of the motors controlling the pumps as well as the sizesof the orifices in the manifolds, and controls the flow rate so that itis maintained within the desired range.

Along with delivering all of the product to the storage chamber 44, theinvention also contemplates that the device be equipped with a means fordiverting the output stream from chamber 34 to either of two or morechambers or channels, thereby providing a mechanism whereby product maydiverted from the primary flow to the storage chamber 44 if the sensorsdetermine it to be below acceptable quality levels, until the sensorscan shut the device down.

The mixer 33 is also equipped with a determining means 46, which can beone or more detectors that operate by use of a feedback system so thateach respective detector is connected to or configured so that itcontrols the temperature controlling means 16 and 18 and the meteringsystems 26 and 28. One such detector can be an optical sensor fordetermining the optical properties of the mixture within chamber 34 forpurposes of adjusting the temperature and flow rate of the componentsfor optimum product quality. A certain degree of opacity is theindication of successful mixing. If the material develops a transparencyand transmits more light than is expected, this is determined by asimple photoresistor or phototransistor detector circuit, and thecontrolling mechanism is instructed to divert the stream and or shutdown the system. Another detector can be a rheometric device, orviscometer, that determines the viscosity of the mixture for furtherprocess quality monitoring and control. As with the optical sensor, therheometric device would also be able to control the temperaturecontrolling means 16 and 18 and the metering systems 26 and 28, so as toadjust the temperature and flow rate of the components.

The mixing device 20 may be equipped with a means 48 of applyingultrasonic energy to the pre-mixing chamber 30 and/or the mixing chamber34. Use of ultrasonic energy in the formation of lipid vesicles isdescribed, for example, in Gersonde, et al., U.S. Pat. No. 4,452,747;Huang, Biochemistry 8:344(1969); Papahodjopoulos, et al., BiochimBiophysica Acta 135:624 (1967); Schneider, U.S. Pat. No. 4,089,801; andHsu, U.S. Pat. No. 5,653,996, the disclosures of which are incorporatedherein by reference.

The purpose of the ultrasonic energy is to facilitate formation of adesirable vesicle size and/or size distribution. Ultrasonic energy canbe applied by means of a transducer that is in direct contact withvarious metal surfaces of the pre-mixing chamber. Depending on theplacement of the transducer relative to the point at which the lipidphase and the aqueous phase make contact, the application of ultrasonicenergy can be used to either aid in the formation of multilamellarliposomes or to help convert the multilamellar vesicles into unilamellarvesicles of certain constrained size ranges.

The process by which large unilamellar and multilamellar vesicles may beconverted to smaller multilamellar and or unilamellar vesicles isdescribed by Gregoriadis (CRC Press), supra. The multilamellar liposomesproduced by the method and apparatus of the invention are typicallywithin the range of 10 nm to 100 μm, and are preferably about 0.2 to 25μm, more preferably about 0.5 to 20 μm in diameter. Preferably the smallunilamellar liposomes produced by the method and apparatus of theinvention are about 20 to 200 nm in diameter, while the largerunilamellar liposomes produced by the method and apparatus of theinvention are about 200 nm to 2 μm in diameter.

In addition, application of ultrasound also serves to induce moreuniformity in the size and distribution of the vesicles, relative to thefrequency at which the ultrasonic energy is being transmitted. Suitablemeans for accomplishing this include any means for generatinghigh-frequency electric energy, such as generators, sonicators,ultrasonic homogenizers and tissue disruptors. For example, highfrequency electric energy is supplied from an electric generator, thenconverted into ultrasonic energy or vibrations and transmitted by anultrasonic transmitter into one or both of the mixing chambers.

The mixing device 20 is also equipped with a means 50 for controllingthe temperature of the apparatus, in particular for controlling thetemperature of the open space surrounding the various components of theapparatus such as the transfer means, pre-mixing system and mixer. Thetemperature controlling means 50 is similar in form and function to thetemperature controlling means 36 described above. It is preferable tomaintain the temperature within the interior of the mixing device withinthe range of about 20 to 80° C., preferably about 50 to 70° C., morepreferably about 60° C., in essence a temperature close to that withinthe mixing chamber 58.

The components of the mixing device 20 can be encased in a sealedcontainer 52, which can replicate a “clean room” such as is necessary inthe production of pharmaceuticals, for example. Container 52 would befeatured with inlet 54 and outlet 56 valves as a method of sterilizingand sanitizing the entire outside surfaces of the components containedtherein such as the metering systems and pre-mixer and mixer componentssuch as their respective chambers, along with the inside surface of thecontainer itself. Steam, ethylene oxide or liquid sterilants aresuitable for use as sterilizing or sanitizing agents to be delivered byinlet valve 54, and subsequently removed by outlet valve 56. The device20 would also be equipped with cleaning means 58 by which the interiorof the components contained therein such as the metering systems andpre-mixing and mixing chambers could be cleaning, such as by theintroduction of heated cleaning agents such as chlorohexidine, sodiumhypochlorite, soaps, detergents, ethanol water mixtures, and so forth.

The apparatus 10 may also be equipped with a heating/cooling means 60,positioned external to the mixing device 20. The dispensing means 40 canbe shaped to extend beyond the periphery of the mixing device and fittedwith such a heating/cooling means, which can be configured as a jacketsurrounding the dispensing means having a cooled or heated fluid flowingwithin.

A slight modification of the schematic illustration of FIG. 1 providesanother embodiment of the apparatus of the invention for the productionof liposomes, and is particularly suited for the production of ULVs.After the formulation is mixed in chamber 34, it is dispensed viadispensing means 40 and control means 42. However, before being directedto a storage chamber or packaging machine, the formulation is subjectedto high pressure in-line homogenization or sonication, thereby causingthe vesicles to be reduced in size or causing them to agglomerate,resulting in a uniform sizing. After this process is complete, theformulation is then directed to the storage chamber or packagingmachine.

One embodiment of the invention is illustrated in FIGS. 2 and 3, whereFIG. 2 is a cut-away side view and FIG. 3 is a top view. The apparatus100 has four main parts: individual means for storing each component ofthe formulation 112 and 114, a sealed container 152, encasing thevarious parts of the mixing device, and an open box 170, which holds thecontrol elements as will be described below.

The larger vessel 112 would typically be used to store a first liquidphase, such as an aqueous phase component and the smaller vessel 114would typically be used to store a second liquid phase, such as a lipidphase component. It is desirable to store each component at a settemperature and this is readily accomplished by means of temperaturecontrols. Temperature control 118, which controls the temperature ofvessel 114, is shown in FIG. 2. Temperature control 118 could be adiscrete thermocouple based controller as is available from OmegaEngineering, Inc., for example, and would be connected to power source172, for example, an electrical outlet.

Each component is delivered to the mixing device 120 by pressurizedtransfer means 122 and 124, each of which is fitted with a precisemetering system 126 and 128 to control the amount of materialtransferred from the storage means to the pre-mixing chamber 130.Metering system 128 is shown in FIG. 2 as comprising a precise meteringpump 174, along with a manifold 176 to control the pump output and inputin order to eliminate any pressure pulsations of the component streamthat would interfere with the precise mixing ratios needed forconsistent product quality. Essentially, the manifold serves to converta pulsed input from the pump to an unpulsed output by means of phasecompensation. The metering system is driven by a motor 178 such as astepper motor.

The mixing device 120 has a pre-mixing system such as a pre-mixer 129having a pre-mixing chamber 130, where the individual components areintroduced under pressure by pressurized transfer means 123 and 125.Accordingly, the pre-mixing chamber has a first inlet orifice 136connected to transfer means 123 for inlet of the aqueous phase, a secondinlet orifice 138 connected to transfer means 125 for inlet of the lipidphase, and an outlet orifice 140 connected to transfer means 132. Thepre-mixer is designed to create a turbulent vortex, whereby onecomponent stream is injected by means of high pressure into a secondcomponent stream or both streams are injected by means of high pressure.

The pre-mixed formulation is then transferred by transfer means 132 andintroduced into a mixer 133 having, for example, a laminar division typeinline mixing chamber 134 of high shear, high pressure design. After theformulation is mixed, it is dispensed via dispensing means into astorage chamber or a means for packaging such as a packaging machine,not shown. In addition; the invention also contemplates use of a meansfor further modification of the properties of the liposomes, which wouldbe positioned immediately after the dispensing means. Such means forfurther modification can be, for example, a means for homogenization orsonication. This would enable one to make a ULV-containing formulationfrom a MLV- or OLV-containing formulation. The modified formulationcould then be dispensed into a storage chamber or a means for packaging,as indicated above for the mixed formulation. However, the modifiedformulation could also be used in a separate apparatus. It could beplaced in a storage vessel and, with its own metering system, be used asa third component in another formulation.

As indicated above, the apparatus 100 includes a box 170 that has anopen end 180 to permit access to the power components, electricalsystems, etc. Along with the temperature control 118, power source 172and motor 178, this box also houses an indexer 186 that controls themotor 178, an indexer controller 182 for controlling the indexer 186,and a relay 184 for controlling the temperature control 118.

FIG. 2 only shows the components that are connected to metering system128. Metering system 126, shown in FIG. 3, has similar components thatrelate to its performance. FIG. 3 illustrates the temperature control188, power source 190, motor 192, and indexer (not shown) that controlsmotor 192, an indexer controller 194, and a relay (not shown) all ofwhich work with metering system 126. There also is a relay (not shown)for controlling the temperature control 150.

Container 152 is sealed by means of a cover or lid 196 that isremovable, but bolted in place, and preferably includes a rubber seal,for example, to provide for a tightly sealed system during operation.The container can also be sealed by numerous means, including by way ofexample and not limitation, clamps, drawing a vacuum in the case ofsensitive ingredients, and by means of a lip and channel configuration.

The mixing device 120 is equipped with a generator 110, as means ofapplying ultrasonic energy to the pre-mixing chamber 130 and/or themixing chamber 134. In the embodiment shown in FIGS. 2 and 3, thegenerator 110 is shown as being mounted on the pre-mixing chamber 130,an appropriate position when ultrasonic energy is being directed to thepre-mixing chamber. It is understood however, that the generator can bemounted on the transfer means 132 if it is desired to direct energy tothe mixing chamber 134. This latter configuration can be in lieu of orin addition to the generator mounted on the pre-mixing chamber.

FIG. 3 provides another view of this embodiment of the invention. Thisview shows that the mixing device is also equipped with a means 150 forcontrolling the temperature of the device, and a power source 198. Alsoshown in FIG. 3, is a top view of metering system 126 which comprises aprecise metering pump 200, along with a manifold 202. The meteringsystem is driven by a motor 194 as noted above.

One skilled in the art will recognize other comparable parts that can beassembled in the manner shown in FIGS. 2 and 3 to produce a liposomalformulation in the manner described herein. In particular, suitableprecise metering pumps are well known in the art. Particularly wellsuited for the method and apparatus of this invention is the Travcyl™metering pump (Encynova International, Inc., Broomfield, Colo.), whichprovides the accurate and efficient fluid delivery required by thisinvention. A simplified version of this pump is shown in FIGS. 4 and 5.

FIG. 4 shows one embodiment of the precision metering system 128 of theinvention, having a precise metering pump 174 and manifold 176. The pumpand manifold are typically provided with a plurality of inlets andoutlets. The manifold has a plurality of inlets and outlets thatcorrespond to the number of outlets and inlets on the pump. In addition,the manifold has an inlet whereby lipid or aqueous phase is transferredfrom the storage vessel and an outlet whereby the formulation componentsis transferred to the pre-mixing system.

A pump 174 having four inlets and four outlets is illustrated in FIG. 5.The formulation component is transferred from its storage vessel 114 tothe manifold 176 via pressurized transfer means 124 prior to enteringthe pump. The component is transferred to the pump by pump inlet means300, 302, 304 and 306. The component then returns to the manifold 176 bypump outlet means 308, 310, 312 and 314, and then exits the manifold bypressurized transfer means 125 for delivery to the pre-mixing chamber130. The pump inlet and pump outlet means are typically comprised of aflexible tubing material such as Teflon, PVDF, polypropylene, stainlesssteel tubing or armored polymer tubing. Pump inlet 300 and pump outlet308 are both connected to one of four cylinders in pump 174. Similarlyinlet/outlet pairs 302/310, 304/312 and 306/314 are connected to theremaining three cylinders, respectively.

FIG. 6 illustrates one embodiment of a manifold 176 useful incombination with the pump 174 of FIG. 5. Similar to the pump, themanifold is provided with four inlets and four outlets, which arenumbered and identified separately. Each pump outlet means communicateswith a manifold inlet means, and each pump inlet means communicates witha manifold outlet means. In addition, the manifold has an inlet orificethat communicates with the storage vessel and an outlet orifice thatcommunicates with the pre-mixer, as described in detail below.

Referring now to FIG. 6, the formulation component is transferred fromits storage vessel 114 to the manifold via pressurized transfer means124 at manifold inlet orifice 141. The component is then transferred tothe pump by manifold outlet means 324, 326, 328 and 330, where manifoldoutlet 328 is on the back side. The component then returns from the pumpto the manifold by manifold inlet means 316, 318, 320 and 322, wheremanifold inlet 318 is on the back side, and then exits the manifold bypressurized transfer means 125 at manifold outlet orifice 142 fordelivery to the pre-mixing chamber. The manifold can be for example, asolid block in which the four inlet means are laterally bored holes, asillustrated in FIG. 7. The same configuration holds for the four outletmeans. An axially bored hole that meets at the junction of the fourinlet means forms the manifold outlet orifice 142 connecting thepressurized transfer means 125 to the manifold. Similarly, an axiallybored hole that meets at the junction of the four outlet means forms themanifold inlet orifice 141 connecting the pressurized transfer means 124to the manifold. This is illustrated in FIG. 8.

In operation, the two component, i.e., precursor phases provided for inthe formulation are each prepared and heated to the proper temperatures,at which time they are transferred to the apparatus 100. Typically,Phase I of the mixture (the aqueous phase) is prepared and heated and isintroduced into the larger of the two vessels 112, which has beenpreheated to the required temperature before filling. Phase II of themixture (the lipid phase) is prepared and heated, is then introducedinto the smaller vessel 114 which has also been preheated to therequired temperature. Once stored in the pre-heated vessels 112 and 114,the phases are maintained at the required temperature, via individualfeedback and control systems. The apparatus is equipped with two pumps174 and 200, each of which is equipped with four positive pressure,positive displacement pumping chambers.

The four inputs from each pump are combined through the use of uniquemanifolds 176 and 202. The four outputs of each pump are then pumpedthrough the same respective manifold and then transferred in a preciseratio to the pre-mixing chamber 130. The result is a near or virtuallypulse-less flow, which permits of precise metering ratios of the twocomponents, i.e., the lipid and aqueous phases that constitute theliposomal formulation. The two components are each individually pumpedthrough the use of the near “pulse-less” precision metering pumps 174and 200 into the premixing chamber 130 where they are introduced to eachother in a precise ratio. The two phases are then introduced into theinline mixing chamber 134 of appropriate length, diameter andcomposition for the specific product to be manufactured.

The near “pulse-less” action is the pump is achieved by use of the fourpump inlet means, each of which is 90° out of phase with the precedinginlet means and the successive inlet means. For example, referring tothe pump 174 shown in FIG. 5, there are four inlet means shown, 300,302, 304 and 306. The operation curve of inlet means 302, for example,is out of phase by 90° from the operation curve of inlet means 300 and304, and so forth. In operation then, these curves cancel each other outto provide for continuous, near “pulse-less” flow of the component fromthe vessel 114, to the manifold 176 to the pump 174, back to themanifold 176, then on to the pre-mixing chamber 130.

The two phases can be slightly intermixed in an interfacial juncture inthe pre-mixing chamber 130, from which they are then forced throughtransfer means 132 into the static mixer 134. The design of thepre-mixing chamber incorporates surfaces that create specific turbulencepatterns that have very specific effects on the properties of theliposomes during their formation. In addition, various baffles may beadded to the pre-mixing chamber or machined into the inner surface ofthe chamber, to increase turbulence.

The design of the pre-mixing chamber 130 and the mixing device permitthe use of an ultrasonic generator to be used on either or both of thechambers. The frequency and signal power of the generator can becontrolled by the same computer that monitors and controls thetemperatures of the supply vessels, the temperature of the enclosure andthe mixing chambers, and the flow rates of the two precision meteringpumps.

The length and internal components of the mixing chamber 134 areselected so as to maximize the effectiveness of the mixing process, asdescribed above.

The mixer feeds product through a determining means 146 which can be asensing device or detector where the optical properties of the productare measured and the results supplied to a monitoring and processcontrol system, which is responsible for the monitoring and adjustmentof temperature, flow rate and mixing proportions of each of the twophases based on sensor values, programmed responses and operator input.The monitoring and process control system typically involves suitablesensor and readout display devices, and the necessary adjustments can bemade manually by the operator or the entire system can be readilycomputerized by methods as are well known in the art.

Another embodiment of the invention is illustrated in FIGS. 9 and 10,where FIG. 9 is a cut-away side view and FIG. 10 is a top view. Thisembodiment illustrates some variations on the embodiment of FIGS. 2 and3, and it is understood that the invention encompasses othercombinations of these two embodiments. In particular, the embodiment ofFIGS. 9 and 10 illustrate one means by which temperature of the storagevessels can be monitored and maintained, an alternate manifold and analternate pre-mixing system for the aqueous and lipid phases, along withillustrating one embodiment of means by which the entire operation canbe run, monitored and controlled.

Referring now to FIGS. 9 and 10, the apparatus 400 is equipped withindividual means for storing each component of the formulation 402(larger vessel to store the aqueous phase components) and 404 (smallervessel to store the lipid phase component), and a sealed container 406,encasing the various parts of the mixing device 415 and the controlelements.

The temperature of each storage vessel is controlled by individualtemperature sensors 408 and 410, along with heating elements 412 and414. Each temperature sensor is inserted into the fluid contents of itsrespective vessel. One preferred type of temperature sensor is aplatinum resistance temperature sensor device, as such devices are wellsuited to precisely maintaining the temperature of the contents at a settemperature. The heating element is configured to wrap around theexterior of the individual vessel and is typically a thermal foil heatercomprised of a heating element encapsulated within a flexible siliconerubber jacket.

Each component is delivered to the mixing device 415 by pressurizedtransfer means 416 and 418 to individual precise metering systems 420and 422 to control the amount of material transferred from the storagemeans ultimately to the pre-mixing chamber 424, and then to the in-linemixer 426. Precise metering system 420 comprises a manifold 417 and aprecise metering pump 419. The system also includes housing 421, whichcontains a motor, controller and power supply. Similarly, precisemetering system 422 comprises a manifold 423, precise metering pump 425,and similar housing 427. Details of this embodiment of the pre-mixingsystem of the invention will be described in detail below.

The pre-mixing system of this embodiment is best illustrated withreference to FIG. 10. The aqueous phase exits the precise meteringsystem 420 by pressurized transfer means 428, which is illustrated ashaving a straight section 430, an L-fitting section 432, and a T-shapedsection 434, connected by coupler flanges 436, 437, 438 and 439. TheT-shaped section 434 is connected to the pre-mixing chamber 424 bycoupler flange 440 and coupler flange 441 on the pre-mixing chamber,joined with a silicone gasket and clamp. The lipid phase exits theprecise metering system 422 by pressurized transfer means 442, which hasa straight section 444 having a diameter approximating that of thesections of pressurized transfer means 428, and an injector section 446having a smaller diameter than the sections of pressurized transfermeans 428. The sections of pressurized transfer means 428 are attachedto each other and joined to section 434 by means of coupler flange 448.The injector section 446 extends within the T-portion of section 434 andexits directly into the pre-mixing chamber 424.

In operation, the aqueous phase exiting the straight portion of T-shapedsection 434 encounters the exterior length of the injector section 446and turbulence is created before the aqueous phase enters the pre-mixingchamber 424, where it is mixed with the lipid phase entering the chambervia injector section 446. This is illustrated in FIG. 11, which shows apartial cross-section of the pre-mixing system 450. This configurationof the juncture of the aqueous phase pressurized transfer means 428 andthe lipid phase pressurized transfer means 442 illustrates oneembodiment of the invention by which the lipid and aqueous phases can beintroduced in a precise manner and provide for temperature stabilizationdue to concentric flow.

Turbulence is introduced into the aqueous phase within the branchedportion 433 of the T-shaped section 434. The injector section 446.fitssnugly into the recess of the flange 448 of the T-shaped section 434,and prevents backflow of the aqueous phase in to the straight section444 of the lipid phase pressurized transfer means 442. The straightsection 444 is connected to the T-shaped section by coupler flange 443.The recursive flow of the aqueous phase into the chamber 435 of thebranched portion 433 creates a damping effect. The ratio of (a) theinternal cross-sectional area of the branched portion 433 which is notoccluded by injector section 446 to (b) the internal cross-sectionalarea of injector section 446 is determined by the desired volumetricratio of the aqueous phase to the lipid phase for any given formulation.

FIG. 11 also illustrates the cross-section of a suitable configurationof the in-line mixer 426, as having a plurality of baffles 452. However,it is understood that this invention is not limited to thisconfiguration and any suitable configuration of the in-line mixer isencompassed by the invention, as long as turbulent mixing of the aqueousand lipid phases is achieved.

Referring again to FIG. 10, the apparatus 400 is provided with a heater454, such as a finned resistive heater which serves to maintain thetemperature within mixing device 415, more specifically the temperaturesurrounding the pressurized transfer means 428 and 442, the premixingchamber 424 and in-line mixer 426. The apparatus also comprises atemperature sensor 464, which serves to monitor the temperature withincontainer 406 and adjust the heater 454 accordingly.

FIG. 12 illustrates another embodiment of a manifold 423 useful incombination with the pump 425 of FIG. 10. This embodiment of themanifold will be described in relation to the transfer of the lipidphase. However, a similar configuration can be used for manifold 417 andpump 419, as pertains to the transfer of the aqueous phase. Similar tothe pump, the manifold is provided with four inlets and four outlets. Inthe pump/manifold embodiment of FIG. 4, there are transfer meansconnecting the pump inlets/manifold outlets and pump outlets/manifoldinlets. In the embodiment of FIG. 9, the various inlets and outlets arepositioned on adjacent faces of the pump and manifold such that eachpump outlet means communicates directly with a manifold inlet means, andeach pump inlet means communicates directly with a manifold outletmeans. In addition, the manifold has an inlet orifice that communicateswith the storage vessel and an outlet orifice that communicates with thepre-mixing system, as described in detail below.

As with the pump in FIG. 5, the near “pulse-less” action is the pump isachieved by use of the four pump inlet means, each of which is 90° outof phase with the preceding inlet means and the successive inlet means.For example, referring to pump 425 shown in FIG. 13, there are fourinlet means shown, 350, 352, 354 and 356. The operation curve of inletmeans 352, for example, is out of phase by 90° from the operation curveof inlet means 352 and 356, and so forth. In operation then, thesecurves cancel each other out to provide for continuous, near“pulse-less” flow of the component from the vessel, to the manifold 423to the pump 425, back to the manifold 423, then on to the pre-mixingchamber 424.

Referring now to FIG. 12, the lipid phase is transferred from itsstorage vessel 404 to the manifold 423 via pressurized transfer means418 and manifold inlet orifice 332. The lipid phase is then transferredto the pump 425 by manifold outlet means 334, 336, 338 and 340, all ofwhich are located on back face 342 of the manifold. FIG. 14 illustratesthe back 342 of the manifold 423, which has the same configuration asthe front face 344 of the precise metering pump 425, as shown in FIG.13. The manifold and pump fit tightly together by means of an O-ring anda plurality of fastening means, which can be bolts for example. Thebolts fit through a plurality of bored holes 346 that extend through themanifold 423 and match up with a plurality of bored holes 348 located onthe face of the pump 425. The pump is fitted with four inlet means, 350,352, 354 and 356 that communicate with manifold outlet means 334, 336,338 and 340. As with the embodiment of FIG. 4, the pump has fourpositive pressure, positive displacement pumping chambers or cylinders,not shown. Pump inlet 350 and outlet 358 communicate with one cylinder,inlet 352 and outlet 360 communicate with another cylinder, and soforth.

The lipid phase then exits the pump through pump outlet means 358, 360,362 and 364, which communicate with manifold inlet means 366, 368, 370and 372. The lipid phase subsequently exits the manifold throughmanifold outlet orifice 374 located on the front face 376 of themanifold. Manifold outlet orifice 374 communicates with pressurizedtransfer means 442.

The manifold 423 is manufactured as a solid block of metal such asstainless steel or a suitable alloy such as the nickel alloy Hastelloy™.This manifold is configured as a square or rectangular box and, as shownin FIG. 12, has six sides or faces, which are identified as the frontface 376, the back face 342, the top face 378, the bottom face 380, afirst side face 382 and a second side face 384. The manifold is alsoconfigured to have two lateral planes, each of which marks the locationof a set of independent flow channels. The two planes are bestillustrated by reference to FIGS. 15 and 16, which are cross-sectionalviews of the manifold of FIG. 12, taken along lines 15—15 and 16—16,respectively.

FIG. 15 illustrates a first lateral plane. A first and second set oftransverse flow channels, 385 and 386 are positioned so as to intersectto form an “X”. The first flow channel 385 is formed by boring an angledhole from side face 384 to a point some distance before side face 382such that the channel 385 does not go through the entire manifold. Theend of channel 385 is then plugged with plug 387, which prevents flowout of the manifold and can be made of threaded Teflon®, rubber, plasticand any other suitable materials that can be configured to fit securelyin the channel. The second flow channel 386 is formed by boring anangled hole from the top face 378 to a point some distance before bottomface 380 such that the channel 386 does not go through the entiremanifold. The end of channel 386 is left open to form manifold inletorifice 332.

Referring now to FIG. 16, the second lateral plane is illustrated. Afirst and second set of transverse flow channels, 388 and 389 arepositioned so as to intersect to form an “X”. The first flow channel 388is formed by boring an angled hole from side face 382 to a point somedistance before side face 384 such that the channel 388 does not gothrough the entire manifold. The end of channel 388 is then plugged withplug 390. The second flow channel 389 is formed by boring an angled holefrom the bottom face 380 to a point some distance before top face 378such that the channel 389 does not go through the entire manifold. Theend of channel 389 is then plugged with plug 391. It is important tounderstand that the flow channels in one lateral plane are independentfrom and do not communicate with the flow channels in the other lateralplane, i.e., there is no fluid flow between the channels 385, 386 inFIG. 15 and channels 388, 389 in FIG. 16.

After the flow channels are bored into each lateral plane, a pluralityof manifold inlet and outlet means are bored into the manifold to apre-specified depth. Manifold inlet means 366, 368, 370 and 372 aredrilled in the back face 342 to a depth sufficient to meet with andcommunicate with flow channels 388 and 389 in the lateral plane shown inFIG. 16. FIG. 17, which is a cross-sectional view of the manifold takenalong line 17—17 in FIG. 12, illustrates this even further. It can beseen that manifold inlet means 370 is bored at a depth to meet with flowchannel 389. Manifold outlet means 334, 336, 338 and 340 are drilled inthe back face 342 to a depth sufficient to meet with and communicatewith flow channels 385 and 386 in the lateral plane shown in FIG. 15.FIG. 17, which is a cross-sectional view of the manifold taken alongline 17—17 in FIG. 12, illustrates this even further. It can be seenthat manifold inlet means 338 is bored at a depth to meet with flowchannel 386. After the manifold inlet and outlet means are bored, themanifold outlet orifice 374 is bored in the face 376 to a depthsufficient to meet with and communicate with flow channel 389, as shownin FIG. 17.

In operation, the lipid phase enters the manifold 423 though themanifold inlet orifice 332. It flows through flow channels 385 and 386,and then exits the flow channels by means of manifold outlet means 334,336, 338 and 340, which communicate with pump inlet means 350, 352, 354and 356, respectively. Fluid moves through pump inlet 350 in to one ofthe pump cylinders then exits via outlet means 358, and so forth withall four inlets and outlets of the pump, such that the lipid phase exitsthe pump 425 as a pulsed stream by means of pump outlet means 358, 360,362 and 364, which communicate with manifold inlet means 366, 368, 370and 372, respectively. The lipid phase then enters and flows throughflow channels 388 and 389, and exits the manifold through the manifoldoutlet orifice 374.

By the configuration of the manifold and the near “pulse-less” action ofthe pump, which uses a plurality of inlet and outlet means, each ofwhich are 90° out of phase, a pulsed input is delivered to thepre-mixing system as a virtually pulse-less flow of lipid phase oraqueous phase.

FIGS. 18-20 illustrate typical embodiments of a monitoring and processcontrol system useful for the operation of the apparatus describedherein, and are exemplified for the apparatus 400 of FIG. 10. FIGS. 18and 19 illustrate control panels for the invention that are fitted witha plurality of connectors, indicator lights and controllers, which areconnected by appropriate wiring (not shown) to the parts of theapparatus that they control, monitor or provide power to.

FIG. 18 illustrates a first control panel 456. Temperature sensorconnector 458 is attached to the lipid phase resistance temperaturedevice 410, while power connector 460 provides power to the lipid phaseheating element 414. Data communication terminal 462 is attached to acomputer 466, for example by means of a standard 9-pin serial port.Indicator light 468 indicates whether the heating elements 412 and 414and heater 454 are on or off. Similarly, indicator light 470 indicateswhen the controllers (as described in FIG. 19) and precise meteringsystems 420 and 422 are on or off.

FIG. 19 illustrates a second control panel 472. Temperature sensorconnector 474 is attached to the aqueous phase resistance temperaturedevice 408, while power connector 476 provides power to the aqueousphase heating element 412. Power connector 478 is attached to a remotecommunication port (not shown) that requires an interface card orconverter. The remote communication port serves as an interface betweenthe controllers 480, 482 and 484, and the computer that is used tointeractively monitor, control, and record the operation of the deviceand its components systems. Controllers 480, 482 and 484 serve tocontrol heating element 414, heating element 412, and heater 454,respectively.

FIG. 20 is the bottom view of a power distribution module 486, which isfitted with a heat exhaust 488. The module has an on-off switch 490 andpower fuse 492 for the heating elements 414, 412 and heater 454. Themodule also is fitted with an on-off switch 500 and power fuse 494 forthe controllers 480, 482 and 484, and precise metering systems 420 and422. In addition, there is a heater relay cooling fan 496 and main powerinput connector 498.

The instant invention also contemplates methods of producing lipidvesicles using a continuous in-line mixing system. In one embodiment ofthe invention, the method involves first preparing a lipid phase,optionally containing an active agent, and storing the lipid phase in afirst storage means that is maintained at a set temperature, typicallywithin the range of about 20 to 80° C. Similarly, an aqueous phase isprepared and stored in a second storage means that is maintained at aset temperature, typically also within the range of about 20 to 80° C.In one embodiment of the invention, the first and second storage meansare continuously replenished with the lipid and aqueous phases,respectively.

The lipid and aqueous phases are then combined by means of a mixingdevice that has a first and a second metering system, a pre-mixer and amixer. The mixer is typically maintained at a temperature within therange of about 20 to 80° C.

The lipid phase is transferred from the first storage means to the firstmetering system by a first pressurized transfer means and the aqueousphase is transferred from the second storage means to the secondmetering system by a second pressurized transfer means. The lipid phaseis then transferred from the first metering system to a first inletorifice in the pre-mixing system by a third pressurized transfer means.Similarly, the aqueous phase is transferred from the second meteringsystem to a second inlet orifice in the pre-mixing system by a fourthpressurized transfer means. In operation of one embodiment of theinvention, the lipid phase is transferred by the first and thirdpressurized transfer at a fluid flow rate of about 4 to 80 cm³/sec, andthe aqueous phase is transferred by the second and fourth pressurizedtransfer means at a fluid flow rate of about 10 to 100 cm³/sec.

The lipid phase and aqueous phases are transferred to the pre-mixingsystem with a high velocity, which creates turbulent flow. Preferably,the lipid phase and aqueous phases are transferred to the pre-mixingsystem in a precise ratio. In another preferred embodiment, the lipidphase and aqueous phases are transferred to the pre-mixing system in anear pulse-less flow. The lipid and aqueous phases are then combined inthe pre-mixing system by shear mixing under conditions to insure thatthe lipid phase becomes fully hydrated by the aqueous phase to form apre-mixed formulation.

The pre-mixed formulation is then transferred from the outlet orifice ofthe pre-mixing system to the mixer by a fifth pressurized transfer meansor other suitable connection or fitting. A mixed formulation, containinglipid vesicles, is formed in the mixer by causing the pre-mixedformulation to traverse a static mixer. The optical properties of thelipid vesicles are optionally measured. This can be accomplished bymeans of an optical transmission sensing device that uses aphotoresistor or phototransistor, which provides a control signal to acontrolling computer or other process control device, which in turnfunctions to control or adjust the temperatures of the first and secondstorage means, along with controlling the operation of the first andsecond metering systems.

In the final step, the mixed formulation is dispensed from the mixingchamber into a storage chamber, into a means for further modification ofthe properties of the lipid vesicles, or into a means of packaging themixed formulation.

In another embodiment, the method includes a homogenization orsonication step after the dispensing step. This latter embodiment isuseful for the production of unilamellar lipid vesicles. The method mayalso include the addition of a second lipid phase and/or a pre-mixedlipid phase-aqueous phase mixture.

In yet another preferred embodiment of the invention, each meteringsystem in the method described above has a precise metering pump and amanifold. Each pump and manifold has a plurality of inlet and outletmeans, where each pump inlet means communicates with a manifold outletmeans and each pump outlet means communicates with a manifold inletmeans. Each pump inlet means is 90° out of phase with the precedinginlet means and the successive inlet means. Along with a plurality ofinlet and outlet means, the manifold also has a manifold outlet orificeand a manifold inlet orifice. In this embodiment, the method includesthe steps of transferring the lipid phase to the inlet orifice of afirst manifold by the first pressurized transfer means andsimultaneously transferring the aqueous phase to the inlet orifice of asecond manifold by the second pressurized transfer means; transferringthe lipid phase from the plurality of outlet means of the first manifoldto the plurality of inlet means of a first pump and simultaneouslytransferring the aqueous phase from the plurality of outlet means of thesecond manifold to the plurality of inlet means of the second pump;transferring the lipid phase from the plurality of outlet means of thefirst pump to the plurality of inlet means of the first manifold andtransferring the aqueous phase from the plurality of outlet means of thesecond pump to the plurality of inlet means of the second manifold; andtransferring the lipid phase from the outlet orifice of the firstmanifold by the third pressurized transfer means and simultaneouslytransferring the aqueous phase from the outlet of the second manifold bythe fourth pressurized transfer means.

For purposes of illustrating the method of the invention, two componentstreams will be described, a lipid phase and an aqueous phase. However,as described above for the apparatus of the invention, it is understoodthat additional single or mixed phase components streams may be added asdesired. In addition, a stream of liposomes may also be included. Bycombining a liposome phase stream with an aqueous phase stream and lipidphase stream, liposomes in the liposome stream can be furtherencapsulated using the method and apparatus of the invention. One ofskill in the art will readily understand how such additional streams canbe incorporated, for example by an the addition of another storagevessel, metering system, etc., such as is described above.

As noted above, the present invention also pertains to a method for thecontinuous production of a composition of matter, such as lipidvesicles, by in-line mixing. In one embodiment of the invention, themethod comprises: (a) preparing a first phase, such as a lipid phase,and storing the lipid phase in a first storage means that is maintainedat a set temperature; (b) preparing a second phase, such as an aqueousphase, and storing the aqueous phase in a second storage means that ismaintained at a set temperature; (c) combining the lipid and aqueousphases by means of a mixing device having first and second meteringsystems, a pre-mixing system and a mixer, by: transferring the lipidphase from the first storage means to the first metering system by afirst pressurized transfer means and transferring the aqueous phase fromthe second storage means to the second metering system by a secondpressurized transfer means; transferring the lipid phase from the firstmetering system to a first inlet orifice in the pre-mixing system by athird pressurized transfer means and transferring the aqueous phase fromthe second metering system to a second inlet orifice in the pre-mixingsystem by a fourth pressurized transfer means; wherein the lipid phaseand aqueous phases are transferred to the pre-mixing system with a highvelocity creating turbulent flow; combining the lipid and aqueous phasesin the pre-mixing system by shear mixing under conditions to insure thatthe lipid phase becomes fully hydrated by the aqueous phase to form apre-mixed formulation; and transferring the pre-mixed formulation froman outlet orifice of the pre-mixing system to the mixer, such as by afifth pressurized transfer means or other suitable connection orfitting; (d) forming a mixed formulation containing lipid vesicles, inthe mixer by causing the pre-mixed formulation to traverse a staticmixer; (e) optionally measuring the optical properties of the lipidvesicles; and (f) dispensing the mixed formulation from the mixingchamber into a storage chamber, into a means for further modification ofthe properties of the lipid vesicles, or into a means of packaging themixed formulation.

This embodiment is further illustrated by the following discussion.First, the aqueous and lipid phases are prepared and heated to theproper temperatures. An exemplary aqueous phase may be sterile water, aphysiological saline solution, a buffer solution, an aqueouscarbohydrate solution, deuterated water, or other isotopic forms of H₂O,buffered solutions of organic acids and bases, and the like, or anycombination thereof. In addition, the aqueous phase may also contain awater soluble organic solvent such as by way of illustration and notlimitation, polyhydric alcohols including glycerin, propylene glycol,polypropylene glycol, triethylene glycol, polyethylene glycol,diethylene glycol monoethyl ether, etc.; alcohols such as benzylalcohols, etc.; ethers; ketones; esters and glycerin esters such asmonoacetin, diacetin, glycerophosphoric acid, etc.; and various aromaticand aliphatic hydrocarbons including fluorocarbons. Typically theaqueous phase will consist of about 75 to 99wt % sterile water,preferably about 85 to 98 wt %.

An exemplary lipid phase may consist of, by way of illustration and notlimitation, phospholipids such as phosphatidyl chlolines,lysophosphatidyl chlolines, phosphatidyl serines, phosphatidylethanolamines, phosphatidyl inositols, cardiolipin, and sphingomyelin;natural phospholipids such as egg yolk lecithin, soybean lecithin, andsoybean oil based phospholipids; glycolipids, dialkyl-type syntheticsurfactants; polar lipids and neutral lipids; fatty acids; and the like.In addition, the lipid phase may also contain materials such asstearylamine, phosphatidic acid, dicetyl phosphate, tocopherol,cholesterol, lanolin extracts propylene glycol, polyethylene glycol,polypropylene glycol, glycol ethers, ethanol, and the like. Typicallythe lipid phase will consist of about 5 to 20 weight percent (wt %) of aphospholipid, preferably about 8 to 12 wt %. The lipid phase typicallycontains an active agent in the amount of about 0.01 to 35 wt %, morepreferably about 2 to 25 wt %. However, it is understood that lipidvesicles can be manufactured without any active agent contained therein,if desired.

It may be desirable to prepare the aqueous phase in an amount in excessof that needed to produce the desired formulation. This excess allowsfor the stabilization of the temperatures of the manifolds, precisemetering pumps, along with the pre-mixer and in-line mixer assembly.Typically, one may produce the aqueous phase in an amount equal to 1 to3, more typically 1.4 to 2 times the amount required for theformulation. On the other hand, the lipid phase is produced in an amountapproximate to that amount needed to produce the desired formulation.

Once prepared, the lipid phase is placed in one storage means and theaqueous phase is placed in another storage means. These formulations areonly provided to exemplify the methods of the invention and are notintended to be limiting in any manner. It is expected that the methodand apparatus described herein will work with any liposomal formulationthat is desired to be produced, with the same results as describedherein.

This instant invention also relates to lipid vesicles produced by themethod and apparatus described herein. Accordingly, the inventioncontemplates lipid vesicles, either multilamellar, oligolamellar orunilamellar, produced by the various apparatus embodiments describedabove. In a similar manner, the invention contemplates lipid vesicles,either multilamellar, oligolamellar or unilamellar, produced by thevarious method embodiments described above. Any of the aforementionedlipid vesicles can further comprise an active agent encapsulated ineither the aqueous core of the lipid vesicles, within the lipid bilayerof the lipid vesicles, or both. Of particular interest are the activeagents ivermectin and diclofenac.

As recognized by those skilled in the art, while certain materials andprocedures may give better results, the use of particular materials andprocedures are not critical to the invention and optimum conditions canreadily be determined using routine testing. In addition, the inventionalso contemplates the inclusion of additional materials in theformulations to facilitate drug delivery, formulation stability, and soforth. For example, some liposome formulations may acquire a gel-likeconsistency upon cooling to room temperature in the absence of anyadjuvants. Accordingly, conventional thickeners and gelling agents wouldbe added to provide a preparation having the desired consistency fortopical application. Additionally, a preservative or antioxidant oftenwill be added to the preparation.

The amount of active agent to be included in the liposomal preparationis not, per se, critical and can vary within wide limits depending uponthe intended application and the lipid used. The level of the activeagent in the final liposomal formulation of the invention can varywithin the full range employed by those skilled in the art, e.g., fromabout 0.01 to 99.99 wt % of the active agent based on the totalformulation and about 0.01-99.99 wt % carrier, etc. More typically, theactive agent will be present at a level of about 0.01-80 wt %.Preferably, the active agent may be included in an amount of betweenabout 0.01 to 10 wt % of the liposomal preparation and more preferablymay be included in an amount of about 0.01 to 7 wt %.

In employing the active agent-liposome formulation produced with thisinvention for pharmaceutical use, any pharmaceutically acceptable modeof administration can be used. For example, products can be made to suitany accepted systemic or local route of administration such as are wellknown in the art, for example, via parenteral, oral (particularly forinfant formulations), intravenous, nasal, bronchial inhalation (i.e.,aerosol formulation), transdermal or topical routes, in the form ofsolid, semi-solid or liquid or aerosol dosage forms, such as, forexample, tablets, pills, capsules, powders, liquids, lotions, solutions,emulsion, injectables, suspensions, suppositories, aerosols or the like.The formulations produced by the invention can also be manufactured assustained or controlled release dosage forms, including depotinjections, osmotic pumps, pills, transdermal (includingelectrotransport) patches lotions, creams and the like, for theprolonged administration of the active agent at a predetermined rate,preferably in unit dosage forms suitable for single administration ofprecise dosages. The formulations will include a conventionalpharmaceutically acceptable carrier or excipient and the active agentand, in addition, may include other medicinal agents, pharmaceuticalagents, carriers, adjuvants, etc. Carriers can be selected from thevarious oils, including those of petroleum, animal, vegetable orsynthetic origin, for example, peanut oil, soybean oil, mineral oil,sesame oil, and the like. Water, saline, aqueous dextrose, and glycolsare preferred liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients and carriers also include starch,cellulose, talc, glucose, lactose, sucrose, mannitol, gelatin, povidone,malt, rice, flour, chalk, silica gel, magnesium stearate, sodiumstearate, glycerol monostearate, magnesium carbonate, sodium chloride,sodium saccharine, croscarmellose sodium, dried skim milk, glycerol,glycols such as propylene glycol, polyethylene and polypropylene glycolsand their derivatives, esters, salts and combinations of glycols andother fatty alcohols or acids, water, low molecular weight alcohols suchas ethanol, propanol, and the like.

The formulations of the invention also preferably contains anantioxidant such as, by means of illustration and not limitation,tocopherol, more specifically Vitamin E (α-tocopherol), tocopherolderivatives, butylated hydroxyanisole, and butylated hydroxytoluene.Other suitable pharmaceutical carriers and their formulations aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin.

If desired, the liposome formulation may also contain minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, etc.

Another aspect of the invention pertains to an improved method ofproducing compositions of matter such as emulsions, ointments and creamsusing the methods and apparatus described herein. The method andapparatus described above for the production of lipid vesicles arereadily modified by one of skill in the art to produce any of a varietyof other compositions by modifying the starting components and theprocess parameters. Such compositions can also be formulated to includea payload.

Emulsions are two-phase systems in which one liquid is dispersed throughanother liquid in the form of small droplets. When oil is the dispersedphase and an aqueous phase is the continuous phase, the system isdesignated as an oil-in-water (“O/W”) emulsion. Conversely, where wateror an aqueous solution is the dispersed phase and oil or oleaginousmaterial is the continuous phase, the system is designated as awater-in-oil (“W/O”) emulsion. Accordingly, emulsions can readily beproduced by using an aqueous phase, optionally containing a surfactant,and an oil phase, optionally containing various other ingredients andexcipients as a second phase.

Ointments are semisolid preparations that are intended for externalapplication to the skin or mucous membranes. They are generallyrecognized as oleaginous bases containing petrolatum. Such ointments canreadily produced by the method and apparatus described herein using aviscous thixotropic phase as a first phase and a non-viscous oil phaseas a second phase.

Creams are viscous liquid or semisolid emulsions, and can also bedesignated as O/W or W/O. These can be formulated, or example, by usingan emulsion phase and an aqueous phase.

Each of the patent applications, patents, publications, and otherpublished documents mentioned or referred to in this specification isherein incorporated by reference in its entirety, to the same extent asif each individual patent application, patent, publication, and otherpublished document was specifically and individually indicated to beincorporated by reference. While the present invention has beendescribed with reference to the specific embodiments thereof, it shouldbe understood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from the truespirit and scope of the invention and the appended claims. In addition,many modifications may be made to adapt a particular situation,material, composition of matter, process, process step or steps, to theobjective, spirit and scope of the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto.

1. A method for the continuous production of lipid vesicles by in-linemixing, said method comprising: (a) preparing a lipid phase and storingthe lipid phase in a first storage means that is maintained at a settemperature; (b) preparing an aqueous phase and storing the aqueousphase in a second storage means that is maintained at a set temperature;(c) combining the lipid and aqueous phases by means of a mixing devicehaving first and second metering systems, a pre-mixing system and amixer, by: transferring the lipid phase from the first storage means tothe first metering system by a first pressurized transfer means andtransferring the aqueous phase from the second storage means to thesecond metering system by a second pressurized transfer means;transferring the lipid phase from the first metering system to a firstinlet orifice in the pre-mixing system by a third pressurized transfermeans and transferring the aqueous phase from the second metering systemto a second inlet orifice in the pre-mixing system by a fourthpressurized transfer means; wherein the lipid phase and aqueous phasesare transferred to the pre-mixing system with a high velocity creatingturbulent flow; combining the lipid and aqueous phases in the pre-mixingsystem by shear mixing under conditions to insure that the lipid phasebecomes fully hydrated by the aqueous phase to form a pre-mixedformulation; and transferring the pre-mixed formulation from an outletorifice of the pre-mixing system to the mixer; (d) forming a mixedformulation containing lipid vesicles, in the mixer by causing thepre-mixed formulation to traverse the mixer; (e) optionally measuringthe optical properties of the lipid vesicles; and (f) dispensing themixed formulation from the mixer into a storage chamber, into a meansfor further modification of the properties of the lipid vesicles, orinto a means of packaging the mixed formulation; wherein each meteringsystem comprises a precise metering pump and a manifold, where each pumpand manifold have a plurality of inlet and outlet means, each pump inletmeans communicates with a manifold outlet means and each pump outletmeans communicates with a manifold inlet means, where each pump inletmeans is 90° out of phase with the preceding pump inlet means and thesuccessive pump inlet means, and the manifold further comprises amanifold outlet orifice and a manifold inlet orifice.
 2. The method ofclaim 1 wherein said lipid vesicles are multilamellar.
 3. The method ofclaim 1 which further comprises a homogenization or sonication stepafter the dispensing step.
 4. The method of claim 3 wherein said lipidvesicles are unilamellar.
 5. The method of claim 1 wherein the lipidphase comprises an active agent.
 6. The method of claim 1 wherein saidfirst storage means is maintained at a temperature within the range ofabout 20 to 80° C.
 7. The method of claim 1 wherein said second storagemeans is maintained at a temperature within the range of about 20 to 80°C.
 8. The method of claim 1 wherein the step of measuring opticalproperties is by means of an optical transmission sensing device using aphotoresistor or phototransistor, which provides a control signal to acontrolling computer or other process control device.
 9. The method ofclaim 1 which further comprises the addition of a second lipid phase, apre-mixed lipid phase-aqueous phase mixture or a pre-formed lipidvesicle phase.
 10. The method of claim 1 wherein the first and secondstorage means are continuously replenished with the lipid and aqueousphases, respectively.
 11. The method of claim 1 wherein the pressuresare within the range of about 10 to 90 psia.
 12. The method of claim 1wherein the fluid flow rate of the lipid phase is about 3 to 200cm³/sec, and the fluid flow rate of the aqueous phase is about 5 to 300cm³/sec.
 13. The method of claim 1 wherein mixer is maintained at atemperature within the range of about 20 to 80° C.
 14. The method ofclaim 1; the method further comprising: transferring the lipid phase tothe inlet orifice of a first manifold by the first pressurized transfermeans and simultaneously transferring the aqueous phase to the inletorifice of a second manifold by the second pressurized transfer means;transferring the lipid phase from the plurality of outlet means of thefirst manifold to the plurality of inlet means of a first pump andsimultaneously transferring the aqueous phase from the plurality ofoutlet means of the second manifold to the plurality of inlet means ofthe second pump; transferring the lipid phase from the plurality ofoutlet means of the first pump to the plurality of inlet means of thefirst manifold and transferring the aqueous phase from the plurality ofoutlet means of the second pump to the plurality of inlet means of thesecond manifold; and transferring the lipid phase from the outletorifice of the first manifold by the third pressurized transfer meansand simultaneously transferring the aqueous phase from the outlet of thesecond manifold by the fourth pressurized transfer means.
 15. The methodof claim 14 wherein the lipid phase and aqueous phases are transferredto the pre-mixer a precise ratio.
 16. The method of claim 14 wherein thelipid phase and aqueous phases are transferred to the pre-mixer in anear pulse-less flow.
 17. Lipid vesicles produced by the method ofclaim
 1. 18. The lipid vesicles of claim 17 wherein said lipid vesiclesare multilamellar or oligolamellar.
 19. The lipid vesicles of claim 18which further comprise an active agent encapsulated in the aqueous coreof said vesicles, within the lipid bilayer of said vesicles, orencapsulated in the aqueous core and within the lipid bilayer of saidvesicles.
 20. The lipid vesicles of claim 19 wherein said active agentis selected from the group consisting of ivermectin and diclofenac. 21.Lipid vesicles produced by the method of claim
 13. 22. The lipidvesicles of claim 21 wherein said lipid vesicles are unilamellar. 23.The lipid vesicles of claim 22 which further comprise an active agentencapsulated in the aqueous core of said vesicles, within the lipidbilayer of said vesicles, or encapsulated in the aqueous core and withinthe lipid bilayer of said vesicles.
 24. The lipid vesicles of claim 23wherein said active agent is selected from the group consisting ofivermectin and diclofenac.